Study Of The Effects Of Bufalin And Its Derivative BF211 On Proteasome Activities Of A549 Cells And The Possible Mechanisms

Bufalin(BF) is one of the main components of the traditional Chinese medicine ChanSu. BF211 is a derivative of BF. Results of MTT assay showed that the IC50 values of 72 h treatment of BF and BF211 in inhibiting proliferation of A549 cells were 8.15±0.69 nM and 3.72±0.62 nM, respectively. In our previous study, influence of BF/BF211 on protein expression profiles of A549 cells was checked and possible target-related proteins of BF/BF211 were found. Possible signal network of the possible target-related proteins was predicted using bioinformatics analysis. The signal networks of BF and BF211 were similar and suggested that BF/BF211 might have influence on cellular functions such as transcription, translation, mRNA splicing, ribosomal protein synthesis, proteasome function, and etc. Further study confirmed that BF/BF211 could inhibit all three kinds of cellular proteasome activities of cultured A549 cells after 24 h treatment. However, BF/BF211 could not directly inhibit proteasome activities of cell lysate. In the three active subunits of proteasome, only recombinant protein of β1 subunit exhibited weak binding affinity to BF/BF211 in vitro. These results suggested that the effects of BF/BF211 on cellular proteasome activities might be indirect. Results of RT-PCR and Western blotting assay showed that, mRNA and protein levels of β1 subunit were significantly decreased by BF/BF211 treatment. While, mRNA and protein levels of β2 and β5 subunits were not significantly changed by BF/BF211 treatment. Results of negative PAGE analysis showed that the amount of integral 26 S proteasome was decreased in cells treated with BF/BF211. These results suggested that BF/BF211 might cause dysfunction of cellular proteasome by inhibiting expression of proteasome β1 subunit and disturbing assembly of proteasome. Results of knockdown proteasome β1 subunit by siRNA transfection showed that, deficiency in assembly of 26 S proteasome and inhibition of all three kinds of protease activities of cellular proteasome were observed in A549 cells with decreased expression of proteasome β1 subunit. In summary, BF and its derivative BF211 were found in the present study to exhibit inhibiting effects on cellular proteasome activities of A549 cells. Their effects on proteasome activities were indirect and might be related to decrease in expression level of β1 subunit and disturbance in assembly of 26 S proteasome in BF/BF211-treated cells.